Characterization of trehalose lipids produced by a unique environmental isolate bacterium Rhodococcus qingshengii strain FF.

AIMS: The aim of this study was to elucidate the chemical properties and applications of trehalose lipids produced by Rhodococcus qingshengii strain FF and optimize its production yield. METHODS AND RESULTS: Strain FF was identified as R. qingshengii. It was observed to produce biosurfactants in the presence of n-hexadecane. The biosurfactants were identified as the mixture of trehalose triesters and trehalose tetraesters, mainly consisting of TrehC12 C3 C6 C12 :10, TrehC11 C8 C6 :6, TrehC11 C6 C4 :5 and TrehC6 C4 C6 :5 based on the analysis of thin layer chromatography, Fourier transform infrared and flight tandem mass spectrometry. The best carbon source and nitrogen source for producing trehalose lipids was the mixture of n-hexadecane and oleic acid (m : m = 1 : 1) and the organic nitrogen, urea. Under this condition, the production of trehalose lipids could reach 7·97 g l-1 . The crude trehalose lipids showed extremely high surface-active properties and were proven to promote the degradation of naphthalene. CONCLUSIONS: The trehalose lipids produced by R. qingshengii strain FF exhibited high surfactant activity under various conditions and were proven to promote the degradation of naphthalene. SIGNIFICANCE AND IMPACT OF THE STUDY: Rhodococcus qingshengii strain FF is a potential candidate for bioremediation. The trehalose lipids might be used as unique biosurfactants in cosmetic industries, biological formulations and other applications.

Phototherapeutic potential of alternative photosensitizers to porphyrins.

Because of promising clinical results obtained with photodynamic therapy, more and more photosensitizers continue to be isolated (from natural sources), synthesized and evaluated, the development of which is considered to be a key factor for the successful clinical application of photodynamic therapy. Porphyrins and their analogs (as classical types of phototherapeutic agents) have been extensively reviewed. In this review, we have attempted to summarize the phototherapeutic potential (in particular, anticancer and antiviral aspects) of nonporphyrin photosensitizers (as a new generation of phototherapeutic agents) in more detail, which have been relatively much less reviewed hitherto. They include anthraquinones, anthrapyrazoles, perylenequinones, xanthenes, cyanines, acridines, phenoxazines and phenothiazines. They have shown certain phototherapeutic advantages over the presently used porphyrins. Some anthraquinones, perylenequinones, cyanines, phenoxazines and phenothiazines exhibit strong light absorption in the 'phototherapeutic window' (600-1000 nm), high photosensitizing efficacy and low delayed skin photosensitivity. Some of the nonporphyrin photosensitizers (such as rhodamine 123, merocyanine 540 and some cyanine cationic dyes) demonstrate higher selectivity for tumor cells. They can also be explored in connection with selective carcinoma photolysis strategy based on mitochondrion-, lysosome- or DNA-directed localization mode.

A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells.

BACKGROUND: Ratio imaging has received intensive attention in the past few decades. The growing potential of ratio imaging is significantly limited, however, by the lack of appropriate fluorescent probes, for acidic organelles in particular. The classic fluorescent dyes (such as fluoresceins, rhodamines and coumarins) are not suitable for studying acidic organelles (such as lysosomes) because their fluorescence is significantly decreased under neutral or acidic conditions. This has motivated us to develop probes that can be used in ratio imaging that are strongly fluorescent even in acidic media. RESULTS: The compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethyl-aminocarbamoyl) methoxy)phenyl)oxazole (PDMPO) was prepared and characterized as a new acidotropic dual-excitation and dual-emission pH indicator. It emits intense yellow fluorescence at lower pH and gives intense blue fluorescence at higher pH. This unique pH-dependent fluorescence property was readily explored to selectively stain lysosomes and to determine the pH of the organelle in an emission-ratio-imaging mode. PDMPO is selectively localized to lysosomes and exhibits a pH-dependent dual excitation and emission. CONCLUSIONS: PDMPO selectively labels acidic organelles (such as lysosomes) of live cells and the two distinct emission peaks can be used to monitor the pH fluctuations of live cells in ratio measurements. Additionally, the very large Stokes shift and excellent photostability of PDMPO make the compound an ideal fluorescent acidotropic probe. The unique fluorescence properties of PDMPO might give researchers a new tool with which to study acidic organelles of live cells.

Photosensitization with anticancer agents. 17. EPR studies of photodynamic action of hypericin: formation of semiquinone radical and activated oxygen species on illumination.

When hypericin was illuminated with 580 nm light in aqueous solution, the semiquinone radical, singlet oxygen, and superoxide anion radical were detected. The formation of the semiquinone radical and activated oxygen species and the transformation and competition between them depend on the quinone and oxygen concentrations, irradiation time and intensity, and the nature of substrate. In anaerobic solution containing a high concentration of the quinone, the semiquinone radical was predominantly photoproduced. In contrast, in aerobic solution, singlet oxygen is the principal product in the photosensitization of hypericin. Besides singlet oxygen, superoxide anion radical is generated by the quinone on illumination in aerobic solution via the reduction of oxygen by the semiquinone radical, but to a lesser extent than singlet oxygen. The generation of superoxide anion radical is significantly enhanced by the presence of electron donors.

Photosensitization with anticancer agents 19. EPR studies of photodynamic action of calphostin C: formation of semiquinone radical and activated oxygen on illumination with visible light.

When calphostin C was illuminated with visible light, the semiquinone radical, singlet oxygen, and superoxide anion radical were detected. The formation of the semiquinone radical and activated oxygen species and the transformations and competitions between them depend upon the quinone and oxygen concentrations, time and intensity of illumination, and the nature of the substrate. In anaerobic solution, the semiquinone radical was predominantly photoproduced via the self-electron transfer between the excited and ground species. In aerobic solution, singlet oxygen is the principal product in the photosensitization of calphostin C. In addition to singlet oxygen, superoxide anion radical is also generated by the quinones upon illumination in aerobic solution, but to a lesser extent than singlet oxygen. The superoxide anion is produced via the reduction of oxygen by the semiquinone radical, and this process is significantly enhanced by the presence of electron donors.

Photosensitization with anticancer agents. 20--EPR studies on the photodynamic action of phleichrome: formation of semiquinone radical and activated oxygen species on illumination with visible light.

When phleichrome was illuminated with visible light, the semiquinone radical, singlet oxygen, and superoxide anion radical were detected. The formation of the semiquinone radical and activated oxygen species and the transformations and competitions between them depend on quinone and oxygen concentration, duration and intensity of illumination, and the nature of the substrate. In anaerobic solution, the semiquinone radical was predominantly photoproduced via the self-electron transfer between the excited and ground species. In contrast, in aerobic solution, singlet oxygen is the principal product in the photosensitization of phleichrome. In addition to singlet oxygen, superoxide anion radical is also generated by the quinones upon illumination in aerobic solution, but to a lesser extent. The generation of the superoxide anion is significantly enhanced by the presence of electron donors.

Photosensitization by anticancer agents 21: new perylene- and aminonaphthoquinones.

Hypocrellins are under intensive investigation as photosensitizing agents for photodynamic therapy (PDT). A recent advance in the synthesis of hypocrellin congeners resulted in the production of an amino-substituted hypocrellin-B, and its "half chromophore." Both compounds exhibit stronger red light absorption than previously reported hypocrellins, and, therefore, merit investigation as photosensitizers.

Characterization of extracellular chitinolytic activity in biofilms.

Extracellular enzymes produced by bacterial biofilms tend to become an integral, permanent part of the biofilm/substratum system. Thus, characterizing extracellular enzyme activity is an essential component of understanding biofilm ecology. Methods have been presented for characterizing three aspects of extracellular enzyme activity in biofilms: promoter activity of the structural gene, local catalytic activity, and kinetics of collective substrate degradation. The abundance of intracellular transcript derived from a structural gene is only indirectly related to the magnitude of catalytic activity of the corresponding enzyme. This relationship may be particularly tenuous in the case of extracellular enzymes, which must be transported out of the cell in order to become active. Fluorogenic substrates that allow direct detection of an increasingly greater variety of enzyme activities are becoming available. There are technical problems, originating from surface roughness and intrinsic fluorescence, associated with microscopic examination of biofilms on natural materials. Thin films provide one option for acquiring data about biofilms colonizing relevant materials.

Design, synthesis and investigation of mechanisms of action of novel protein kinase C inhibitors: perylenequinonoid pigments.

A series of perylenequinonoid pigments (PQPs) and related compounds were synthesized and screened for the inhibition of protein kinase C (PKC), a key enzyme involved in cellular differentiation and proliferation, and a potential target for anticancer and antiviral chemotherapeutic drugs. This study has established PQPs as efficient PKC inhibitors, and elucidated aspects of the light-enhanced action mode of the PKC inhibitors. Comparative studies between natural and synthetic PQPs led to the recognition of the effect of certain structural features of PQPs on PKC inhibition, including the skeleton of the 3,10-dihydroxy-4,9-perylenequinonoid chromophore and the configuration of the two side chains at positions 1 and 12. Calphostin C was identified as a superior PKC inhibitor of the POP class, and with the latter as a representative structure, we investigated the mechanism of PKC inhibition by PQPs via electron paramagnetic resonance spectroscopy in conjunction with the spin-trapping technique, absorption and fluorescence spectroscopy, photochemical and photobiological studies, and enzyme methodology. Multiple modes of action are suggested for PKC inhibition, comprising the following steps: (1) the binding of PQPs to the PKC regulatory domain via complexation; (2) the photobonding between mercapto groups of PKC cysteine residues and the PQP quinonoid moiety; and (3) the PQP-sensitized photodamage of PKC via Type I and/or Type II photosensitization.

Hypocrellins as photosensitizers for photodynamic therapy: a screening evaluation and pharmacokinetic study.

Hypocrellin compounds were selected as potential photosensitizers for photodynamic therapy (PDT) owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. Parent hypocrellins A and B share an absorption peak at 658 nm. These molecules may therefore be considered useful progenitors of derivatives which absorb more strongly in the red, considering that the ideal sensitizer should absorb in the 650-800 nm range, beyond the absorption range of hemoglobin and melanin, and where light penetration in tissues is maximized through reduced scattering. A series of pure, monomeric hypocrellin derivatives was tested for properties of dark cytotoxicity and photosensitizing potential by clonogenic assay in monolayer cultures of EMT6/Ed murine tumor cells. Their respective toxicities are reported on a molar basis. The in vitro screening assay has, to date, resulted in the selection of four hypocrellin derivatives for further development as photosensitizers for PDT. Cellular uptake for photosensitizing doses of selected compounds was determined by fluorimetry. Dose escalation studies in rodents indicate that potentially photosensitizing doses promote no demonstrable systemic toxicity.

Novel therapeutic and diagnostic applications of hypocrellins and hypericins.

Hypocrellins and hypericins, structurally related plant pigments isolated from Hypocrella bambuase and Hypericum respectively, are known photodynamic agents. This review summarizes certain significant advances in the photophysics, photochemistry and photobiology of these pigments in the last 2 years and discusses their prospects as novel therapeutic and diagnostic agents in the future. Recently, certain unique properties of hypocrellins and hypericins have been explored for a variety of therapeutic and diagnostic applications. In particular, substantial progress has been made in both anticancer and antiviral applications (especially anti-human immunodeficiency virus). The promising anticancer and antiviral results obtained both in vitro and in vivo have led to intensive investigation into their photo-physical and photochemical processes, especially kinetic studies of their intramolecular proton transfer. These compounds offer the potential for a highly sensitive fluorescent redox sensor for investigation of a variety of cellular events. The biomedical advances of hypocrellins and hypericins have been further promoted by significant progress in their chemical synthesis and the recent commercialization of hypocrellins A and B and hypericin.

Antiviral activities of photoactive perylenequinones.

Nine perylenequinones (PQ), including some familiar naturally occurring pigments, were compared for their light-mediated antiviral efficacies. Calphostin C was the most active compound against the two target viruses, herpes simplex virus type 1 and Sindbis virus. Hypocrellins A and B were also very active. However, three cercosporin-like PQ were substantially less active in spite of their high quantum yields of singlet oxygen, whereas phleichrome, another efficient singlet oxygen producer, showed no detectable antiviral activity. One other PQ, which was a very weak singlet oxygen producer, also showed no antiviral activity. None of the active compounds showed significant antiviral activity in the dark. Thus, for some groups of PQ there was correlation between quantum yield of singlet oxygen (1O2) and antiviral efficacy, but there are evidently other structural features of PQ that influence activity.

Uptake kinetics and intracellular localization of hypocrellin photosensitizers for photodynamic therapy: a confocal microscopy study.

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.

Novel method for screening bacterial colonies for phosphatase activity.

Current methods for screening large numbers of bacterial colonies for phosphatase activity, rely heavily on the use of colorimetric assays. While such methods have been applied extensively in the laboratory, they are not without their drawbacks. We here describe a precipitating fluorescent probe that can be used to screen phosphatase activity in bacterial colonies. This probe can be incorporated directly into agar plates used to culture the organisms of interest. The approach offers several advantages over current methodologies including the ability to monitor the development of phosphatase activity with colony development, and the ability to distinguish between activity arising from cell-bound and cell-free enzyme. This enzyme probe was successfully used to detect and isolate phosphatase-producing bacteria from activated sludge.

A fluorogenic substrate for beta-glucuronidase: applications in fluorometric, polyacrylamide gel and histochemical assays.

We have developed a fluorogenic substrate, ELF-97 beta-D-glucuronide, that provides significant advantages over existing substrates in detecting beta-glucuronidase activity. ELF-97 beta-D-glucuronide allows the detection of enzymatic activity in situ, yielding a hydrolytic product that exhibits maximal fluorescence within the physiological pH range. This substrate yields a hydrolytic product that demonstrates a more than 100 nm Stokes shift, which minimizes interference from autofluorescence in plant tissue. With the commercial enzyme, ELF-97 beta-D-glucuronide can detect less than 2 x 10(-4) U/ml of beta-glucuronidase activity in solution, and 5 x 10(-4) U per lane in polyacrylamide gels. Assays using transgenic Arabidopsis, whole leaf extracts of GUS-positive, but not GUS-negative plans, show significant GUS activity upon incubation with ELF-97 beta-D-glucuronide. Furthermore, the ability of this substrate to form insoluble precipitates at the sites of enzymatic activity makes it suitable for in situ localization of GUS activity in tissue samples of higher plants.

Photosensitization with anticancer agents. 15. Perylenequinonoid pigments as potential photodynamic therapeutic agents: formation of semiquinone radicals and reactive oxygen species on illumination.

Visible light illumination of solutions of perylenequinonoid pigments generates the corresponding semiquinone radicals, singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide. In anaerobic solution, the semiquinone radicals are predominantly photoproduced via the self-electron transfer between the excited and ground state species. In aerobic solution, singlet oxygen is the principal product in the photosensitization of perylenequinonoid pigments. The 3,10-dihydroxy-4,9-perylenequinonoid chromophore was shown to be the necessary structural requirement for the generation of singlet oxygen, and the side-chains of the quinones had little effect on the production of singlet oxygen. This conclusion is useful in the development of more efficient photodynamic therapeutic agents than natural perylenequinonoid pigments themselves. Such agents should ideally contain the 3,10-dihydroxy-4,9-perylenequinonoid chromophore to produce singlet oxygen together with appropriate elaborated side-chains to permit the selective localization of the sensitizer in tumor tissue. In addition to singlet oxygen, superoxide anion radical is generated by the perylenequinones on illumination in aerobic solution, but to a lesser extent than singlet oxygen, via the reduction of oxygen by the corresponding semiquinone radicals. This latter process is significantly enhanced by the presence of electron donors.

Photosensitization with anticancer agents. 16. The photo-oxidation of hypocrellin A. A mechanism study using 18O labelling.

Hypocrellin A was selected as a representative structure to investigate the mechanism of photo-oxidation of perylenequinonoid pigments (PQPs). It was found that singlet oxygen plays a key role in the photo-oxidation of PQPs. The following processes were determined to be involved in the photo-oxidation of PQPs using 18O atom labelling. After the PQP is photoexcited, it tautomerizes and dissociates to produce the anion, which reacts with singlet oxygen generated during irradiation, resulting in the formation of an endoperoxide. The endoperoxide then rearranges to form a dioxetane which undergoes cycloreversion to produce the final product, a di-alpha-naphthoquinone. The factors affecting the photo-oxidation of hypocrellin A were also investigated, including the pH of the solution, nature of the solvent, irradiation wavelength and hydration phenomena. Of these factors, the extremely strong effect of pH on the photo-oxidation of hypocrellin A may be used as a basis for the selective localization of hypocrellin A in tumor tissue. On the basis of the proposed mechanism for the photo-oxidation of PQPs, it may be possible to design and synthesize an optimal tumor-selective PQP photosensitizer for the photodynamic therapy of human tumors.

Electronic spectra of hypocrellin A, B and their derivatives.

In this paper, the electronic spectra of hypocrellin A and B (HA and HB) are studied in detail. It has been proved that their three visible absorption bands come respectively from the pi pi transition of their conjugated systems and intramolecular proton transfer. In dilute solutions, their fluorescence spectra consist of the fluorescence peak of the neutral monomolecule and that of the zwitter-ions that are formed through proton transfer of excited states. In concentrated solution, the longer wavelength emission band is composed of the overlapped fluorescence peaks of zwitter-ions and excimers. The fluorescence spectra of the crystalline hypocrellin A and B consist of the fluorescence peaks of zwitter-ions and excimers and the fluorescence of neutral monomolecules could could not be observed. Their relative intensities are closely related to the excitation wave-length.

Photosensitization with anticancer agents. 18. Perylenequinonoid pigments as potential photodynamic therapeutic agents: preparation and photodynamic properties of amino-substituted hypocrellin derivatives.

Perylenequinonoid pigments have demonstrated several photodynamic therapeutic advantages over the commonly used hematoporphyrin derivatives. Certain new amino-substituted hypocrellin B derivatives have been prepared, and both direct and reductive aminations of hypocrellin B are described. The photophysical and photochemical properties of the amino-substituted hypocrellin B derivatives were investigated, and their significantly enhanced red absorptivities and strong 1O2-generating functions qualify them as promising photodynamic therapeutic agents. In addition, the superoxide anion radical was also determined to be implicated in the photosensitization of the amino-substituted hypocrellin B derivatives.